G-protein activated K+ channels (GIRK; Kir3) play an important role in the cardiac and brain function, mediating the inhibitory action of many neurotransmitters. GIRK channels are activated by direct binding G-Beta and G-Alpha subunits of G-proteins also directly interact with GIRK and play an important role in determining the specificity of signaling. Over the last years, we have presented evidence that Galpha subunits affect GIRK gating directly and indirectly. However, much of the phenomenology, the physiological impact, and the mechanisms of interaction of Galpha with GIRK remains unclear. These issues are in the focus of the proposed study. Our long-term goal is to understand the molecular mechanisms and the physiological significance of GIRK modulation by Galpha subunits, and to utilize GIRK to study the general problem of specificity and G protein effector interactions in G protein pathways. The specific aims are: (1) Further study of the roles and mechanisms of actions of Ga in GIRK gating. By monitoring protein-protein interactions in vitro and by using physiological assays in Xenopus oocytes and HEK cells, mutagenesis, immunocytochemistry, and fluorescence energy transfer (FRET), we will: i) verify the existence of GcdGIRK interactions and their impact on GIRK activity; ii) clarify which form(s) of Gm is physiologically important; iii) map the sites of interaction between Galpha and GIRK, and study the role of these sites in channel gating and regulation; iv) investigate how GIRK gating is affected by Galpha (2) Understanding of the role of Galpha-GIRK interactions in determining specificity of signaling in this pathway. The physical interactions between the GIRK channel and various Galpha subunits will be monitored, and correlated with the effects of coexpression of these Galpha subunits, on the function of GIRK channels. (3). Study of the physiological correlates of modulation of GIRK by Ga. The roles of GIRK modulation by Galpha in cultured atrial myocytes and neurons will be addressed using electrophysiological methods, by co-expression of proteins, addition of purified proteins and peptides to excised patches, and by knock-down of endogenous G protein subunits. Colocalization and complex formation between Galpha and GIRK will be explored in atrium and brain using biochemical and immunohistochemical methods.